Norepinephrine directly activates adult hippocampal precursors via beta3-adrenergic receptors

Abstract

Adult hippocampal neurogenesis is a critical form of cellular plasticity that is greatly influenced by neural activity. Among the neurotransmitters that are widely implicated in regulating this process are serotonin and norepinephrine, levels of which are modulated by stress, depression and clinical antidepressants. However, studies to date have failed to address a direct role for either neurotransmitter in regulating hippocampal precursor activity. Here we show that norepinephrine but not serotonin directly activates self-renewing and multipotent neural precursors, including stem cells, from the hippocampus of adult mice. Mechanistically, we provide evidence that beta(3)-adrenergic receptors, which are preferentially expressed on a Hes5-expressing precursor population in the subgranular zone (SGZ), mediate this norepinephrine-dependent activation. Moreover, intrahippocampal injection of a selective beta(3)-adrenergic receptor agonist in vivo increases the number of proliferating cells in the SGZ. Similarly, systemic injection of the beta-adrenergic receptor agonist isoproterenol not only results in enhancement of proliferation in the SGZ but also leads to an increase in the percentage of nestin/glial fibrillary acidic protein double-positive neural precursors in vivo. Finally, using a novel ex vivo "slice-sphere" assay that maintains an intact neurogenic niche, we demonstrate that antidepressants that selectively block the reuptake of norepinephrine, but not serotonin, robustly increase hippocampal precursor activity via beta-adrenergic receptors. These findings suggest that the activation of neurogenic precursors and stem cells via beta(3)-adrenergic receptors could be a potent mechanism to increase neuronal production, providing a putative target for the development of novel antidepressants.

Figure 1.

Norepinephrine but not serotonin activates a precursor cell population from the adult hippocampus. A–C, Treatment of adult hippocampal cells with NE but not serotonin (5-HT) in the presence of EGF and bFGF significantly enhanced neurosphere formation with up to a twofold increase observed at 10 μm (mean ± SEM; ***p < 0.001) (A). In addition, norepinephrine treatment generated a number of very large neurospheres, an example of which is shown in C, compared with smaller neurospheres generated in the control (B). Scale bars, 100 μm. D, Neurospheres obtained in the presence of 10 μm norepinephrine were significantly larger than the control neurospheres. Note an increase in the percentage of norepinephrine-derived neurospheres measuring 100–150 μm, 150–200 μm, and >200 μm, but a reduction in the smaller neurospheres measuring <100 μm compared with the control neurospheres (mean ± SEM; *p < 0.05, **p < 0.01***p < 0.001).

Figure 2.

Hippocampal precursors activated by norepinephrine are self-renewing and multipotent. A, A large increase in cell numbers was observed when a single norepinephrine-derived large neurosphere was passaged up to 10 times. B, Relative percentage of the primary neurospheres expressing markers of astrocytes, neurons and oligodendrocytes in control versus NE-treated cultures. Note that all neurospheres examined contained GFAP-positive astrocytes. However, a significantly larger proportion of neurospheres expressed the neuronal marker, βIII tubulin, in the norepinephrine-treated vs the control group. MBP-positive oligodendrocytes were only present in norepinephrine-stimulated neurospheres. C, D, An example of control (C) and norepinephrine-derived (D) neurospheres showing immunofluorescence for GFAP (green) and βIII tubulin (red). Nuclei were stained with DAPI (blue). Scale bars, 100 μm. Note the presence of a large number of βIII tubulin-positive neurons in the norepinephrine-derived sphere. E, MBP-expressing oligodendrocytes (green) were also present in norepinephrine-stimulated neurospheres. Scale bar, 30 μm.

Figure 3.

Norepinephrine and KCl activate different populations of hippocampal precursors. A, Culturing adult hippocampal cells in the presence of NE and KCl resulted in over a 4.5-fold increase in neurosphere numbers compared with a twofold increase in the number of neurospheres observed in the presence of either norepinephrine or KCl alone (mean ± SEM; *p < 0.05). B, Distribution of neurospheres according to size showing approximately a fivefold increase in the number of large neurospheres, measuring >200 μm, obtained in the presence of NE+KCl (mean ± SEM; *p < 0.05, **p < 0.01).

Figure 4.

Hes5-GFP-positive cells coexpress markers of stem cells in the adult dentate gyrus. A, Hes5-GFP-positive cells are predominantly present along the subgranular zone and extend radial-glia like processes through the granule cell layer (GCL) in the adult dentate gyrus. B–D′, Hes5-GFP-positive cells coexpress markers of stem cells such as GFAP (red; B) and nestin (red; C). The coexpression is seen predominantly along the processes of the Hes5-GFP-positive cells (arrowheads; B′, C′). No coexpression was seen with doublecortin (red; D), a marker of newly born neurons. However, doublecortin-positive cells were mainly found in juxtaposition with Hes5-GFP-positive cells (arrowheads; D′). Nuclei were labeled with DAPI (blue). Scale bars, 100 μm.

Figure 5.

Norepinephrine activates a Hes5-expressing precursor population. A, Hes5-GFP- positive and -negative cells were sorted using flow cytometry based on their GFP expression. FSC, Forward scatter. B, Reverse transcriptase-PCR analysis revealed the presence of Hes5 mRNA only in the GFP-positive population. C, The Hes5-GFP-positive population contained all the neurosphere-forming cells. Note that in the presence of norepinephrine almost twice as many neurospheres were obtained from the Hes5-GFP-positive population. No neurospheres were generated from the Hes5-GFP-negative population (mean ± SEM; ***p < 0.001).

Figure 6.

β₃ receptors are expressed on neural precursors, and mediate the norepinephrine-dependent activation. A, Neither the α₁-adrenergic receptor antagonist prazosin nor the α₂-adrenergic receptor blocker yohimbine had any effect on the norepinephrine-stimulated increase in neurosphere numbers. Only the β-adrenergic receptor blocker, propranolol, completely inhibited the norepinephrine-stimulated increase in neurosphere numbers. Note that treatment with propranolol alone had no toxic effect on neurosphere production. A slight but significant increase in the number of neurospheres was also observed in the presence of yohimbine alone. B, The selective β₃ blocker SR59230A completely inhibited the norepinephrine-mediated increase in neurosphere numbers. In contrast, the β₁ receptor antagonist CGP20712 had no effect, whereas the β₂ receptor antagonist ICI118,551 significantly increased neurosphere numbers both in the presence and absence of norepinephrine. C, Expression of β-adrenergic receptors in the sorted population of Hes5-GFP-positive and -negative cells by reverse transcriptase-PCR showed the presence of the β₃-adrenergic receptor exclusively in the Hes5-positive population, whereas β₁- and β₂-adrenergic receptor transcripts were expressed predominantly in the Hes5-negative population. Note that a small amount of β₂ receptor mRNA was also detected in the Hes5-positive population. D, A similar increase in neurosphere numbers was observed in the presence of a selective β₃-adrenergic receptor agonist BRL37344 at 1 and 10 μm, compared with treatment with norepinephrine (mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 7.

Stimulation of β₃-adrenergic receptors increases proliferation of hippocampal precursors in vivo. A–C, Bilateral intrahippocampal microinfusion was verified on Nissl-stained sections. A, A representative coronal section showing the injection track terminating in the hilus region of the hippocampus (Paxinos and Franklin, 2001). The hilus from one hemisphere received a 0.5 μl injection of 10 μm BRL37344 with the contralateral hemisphere receiving a control injection of 0.9% saline. B, C, Nissl-stained sections showing the most ventral point of the microinfusion track (arrows) following infusion of 0.9% saline (B) or BRL37344 (C). Scale bars, 200 μm. D, E, A representative micrograph showing BrdU-labeled cells along the SGZ in saline-treated (D) versus BRL37344-treated (E) hippocampus. The granule cell layer is delineated by the dashed lines. Scale bars, 200 μm. F, A confocal section showing colabeling of a nestin-GFP-positive cell (green) with GFAP (red) in the SGZ of isoproterenol-treated mice. Scale bar, 10 μm.

Figure 8.

Direct application of norepinephrine but not serotonin enhances hippocampal precursor activity in the slice-sphere assay. A, The hippocampus from a postnatal day 7 Wistar rat was dissected and cut transversely into 300 μm slices. The slices were placed on a 0.4 μm membrane filter that was bathed in 1 ml of complete serum-free NeuroCult medium in a 6-well plate. Four filters, each containing 6–7 slices, were generated from a single animal. B, The organotypic slices were cultured at a liquid-air interphase for a period of 6 d. Antidepressants or neurotransmitters were added to the medium on day 1 and half the medium was replaced with fresh medium every alternate day. C, On the sixth day, the hippocampal slices were enzymatically dissociated and cells were plated in a 96-well plate and cultured in the presence of EGF and bFGF to obtain neurospheres. D, The number of neurospheres generated was quantified after 10–12 d in culture, this being representative of the number of proliferating hippocampal precursors present in the slices. E, Serotonin (5-HT) treatment had no effect on the frequency of neurosphere formation either at 10 or 100 μm. However, direct application of NE to the slices resulted in an ∼2-fold increase in the neurosphere frequency at 1 and 10 μm, and a 3.5-fold increase at 100 μm (mean ± SEM; **p < 0.01; ***p < 0.001).

Figure 9.

NRIs but not SSRIs increase the activity of hippocampal precursors in the slice-sphere assay. A, Slices treated with the SSRIs fluoxetine (1 μm) or citalopram (10 and 100 μm) showed no significant change in the frequency of neurosphere generation compared with the untreated slices (control). Treatment with 10 μm fluoxetine decreased neurosphere frequency. B, Reboxetine, a prototypical NRI, significantly enhanced the frequency of neurosphere formation at 10 and 100 μm. Treatment of slices with atomoxetine and maprotiline also resulted in a significant increase in neurosphere frequency. C, Blockade of β-adrenergic receptors by propranolol (10 μm) abolished both the norepinephrine- and the reboxetine-mediated increase in neurosphere frequency (mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001).